1,3-Dihydroimidazole-2-Thione Derivatives for Use in the Treatment of Pulmonary Arterial Hypertension and Lung Injury

ABSTRACT

The present invention relates to compounds of formula I: 
     
       
         
         
             
             
         
       
     
     for use in treating pulmonary arterial hypertension and associated conditions, where R 1 , R 2  and R 3  are the same or different and signify hydrogens, halogens, alkyl, alkylaryl, alkyloxy, hydroxy, nitro, amino, alkylcarbonylamino, alkylamino or dialkylamino group; R 4  signifies hydrogen, alkyl, -alkylaryl or -alkylheteroaryl; X signifies CH 2 , oxygen atom or sulphur atom; n is 1, 2 or 3, with the proviso that when n is 1, X is not CH 2 ; and the individual (R)- and (S)-enantiomers or mixtures of enantiomers and pharmaceutically acceptable salts thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of and claims priority to U.S. patentapplication Ser. No. 15/432,616 filed Feb. 14, 2017, published as U.S.Patent Application Publication No. US 2017-0369477 A1, which is acontinuation of and claims priority to U.S. patent application Ser. No.14/441,043 filed May 6, 2015, now U.S. Pat. No. 9,604,970, which is afiling under 35 U.S.C. 371 of International Application No.PCT/PT2013/000065 filed Nov. 14, 2013, all of which entitled“1,3-Dihydroimidazole-2-Thione Derivatives For Use In The Treatment OfPulmonary Arterial Hypertension And Lung Injury,” which claims priorityto U.S. Provisional Application No. 61/726,119 filed Nov. 14, 2012,entitled, “Therapy,” each of which applications is incorporated byreference herein in its entirety.

FIELD OF THE INVENTION

The present invention relates to new therapeutic applications involvingthe following class of compounds of formula I:

where R₁, R₂ and R₃ are the same or different and signify hydrogens,halogens, alkyl, alkylaryl, alkyloxy, hydroxy, nitro, amino,alkylcarbonylamino, alkylamino or dialkylamino group; R₄ signifieshydrogen, alkyl, -alkylaryl or -alkylheteroaryl; X signifies CH₂, oxygenatom or sulphur atom; n is 1, 2 or 3, with the proviso that when n is 1,X is not CH₂; and the individual (R)- and (S)-enantiomers or mixtures ofenantiomers and pharmaceutically acceptable salts thereof.

SUMMARY OF THE INVENTION

According to a first aspect of the present invention, there is aprovided a compound of formula I:

for use in treating pulmonary arterial hypertension (PAH), where R₁, R₂and R₃ are the same or different and signify hydrogens, halogens, alkyl,alkylaryl, alkyloxy, hydroxy, nitro, amino, alkylcarbonylamino,alkylamino or dialkylamino group; R₄ signifies hydrogen, alkyl,-alkylaryl or -alkylheteroaryl; X signifies CH₂, oxygen atom or sulphuratom; n is 1, 2 or 3, with the proviso that when n is 1, X is not CH₂;and the compound of formula I includes its individual (R)- and(S)-enantiomers or mixtures of enantiomers and pharmaceuticallyacceptable salts thereof. The compound of formula I may be used alone orin combination with another active pharmaceutical ingredient.

In an embodiment, X is O.

In another embodiment, at least one of R₁, R₂ and R₃ is fluorine.Preferably, at least two of R₁, R₂ and R₃ are fluorine and the other ishydrogen.

Unless stated otherwise, in this specification the term alkyl (whetherused on its own or used in combination with other moieties) meanshydrocarbon chains, straight or branched, containing from one to sixcarbon atoms, optionally substituted by aryl, alkoxy, halogen,alkoxycarbonyl or hydroxycarbonyl groups; the term aryl (whether used onits own or used in combination with other moieties) means a phenyl ornaphthyl group, optionally substituted by alkyloxy, halogen or nitrogroup; the term halogen means fluorine, chlorine, bromine or iodine; andthe term heteroaryl means heteroaromatic group, wherein the heteroatomsare selected from O, N or S. Moreover, the terms ‘alkoxy’ and ‘alkyloxy’are interchangeable, unless indicated otherwise.

Preferably, the heteroaryl group is represented by an optionallysubstituted aromatic heterocyclic ring system of up to 10 atomscontaining one to four heteroatoms selected from N, O or S. Suitably,the heteroaryl is a heterophenyl group, and the heterophenyl includesfrom 1 to 4 heteroatoms, the or each heteroatom being independentlyselected from O, N or S. Suitably, the heteroaryl is a heteronaphthylgroup, and the heteronaphthyl includes from 1 to 4 heteroatoms, the oreach heteroatom being independently selected from O, N or S. Examples ofpreferred aromatic heterocycles of up to 10 atoms include but are notlimited to benzimidazole, benzofuran, benzothiophene, benzoxazole,benzothiazole, carbazole, cinnoline, furan, imidazole, indole, indazole,isoindole, isoquinoline, isoxazole, isothiazole, oxazole, oxadiazole,oxathiazole, oxathiazolidine, phenazine, phenothiazine, phenoxazine,phthalazine, pteridine, purine, pyran, pyrazine, pyrazole, pyridazine,pyridine, pyrimidine, pyrrole, quinoline, quinoxaline, quinazoline,tetrazole, thiophene, thiadiazole, thiazole, thiopyran, triazine andtriazole in all their isomeric configurations. These heterocycles may beunsubstituted. Alternatively, these heterocycles may be substituted onceor several times by C₁-C₆-alkyl, C₁-C₆-alkoxy, COOH, SO₃H, CONH₂,SO₂NH₂, CONH₂, SO₂NH₂, thiol, hydroxyl, nitro, cyano, fluoro, chloro,bromo, iodo, CF₃ or OCF₃.

In an embodiment, the compound of formula I has the formula IA,

wherein R₁, R₂ and R₃ are the same or different and signify hydrogens,halogens, alkyl, alkylaryl, alkyloxy, hydroxy, nitro, amino,alkylcarbonylamino, alkylamino or dialkylamino group; R₄ signifieshydrogen, alkyl or -alkylaryl group; X signifies CH₂, oxygen atom orsulphur atom; n is 1, 2 or 3, with the proviso that when n is 1, X isnot CH₂. Thus, the present invention provides a compound of formula IAfor use in treating pulmonary arterial hypertension (PAH), wherein thecompound of formula IA includes its (R) or (S) enantiomer, or a mixtureof (R) and (S) enantiomers, and pharmaceutically acceptable salts oresters thereof.

In an embodiment, X is O.

In an embodiment, n is 2 or 3.

In another embodiment, at least one of R₁, R₂ and R₃ is fluorine.

In an embodiment, the compound of formula IA is(R)-5-(2-aminoethyl)-1-(6,8-difluorochroman-3-yl)-1,3-dihydroimidazole-2-thione(compound A) optionally in salt form. The compound A is suitably(R)-5-(2-aminoethyl)-1-(6,8-difluorochroman-3-yl)-1,3-dihydroimidazole-2-thionehydrochloride.

In an embodiment, the compound of formula I has the formula IB

wherein R₁, R₂ and R₃ are the same or different and signify hydrogen,halogen, alkyl, nitro, amino, alkylcarbonylamino, alkylamino ordialkylamino group; R₄ signifies -alkyl-aryl or -alkyl-heteroaryl; Xsignifies CH₂, oxygen atom or sulphur atom; n is 2 or 3. Thus, thepresent invention provides a compound of formula IB for use in treatingpulmonary arterial hypertension (PAH), wherein the compound of formulaIA includes its (R) or (S) enantiomer, or a mixture of the (R) and (S)enantiomer, and pharmaceutically acceptable salts or esters thereof.

In a preferred embodiment n is 2.

In a further preferred embodiment, X is O.

Preferably R₄ signifies —CH₂-aryl or —CH₂-heteroaryl.

In one embodiment, the aryl group of R₄ is unsubstituted.

The aryl group of R₄ may preferably be phenyl.

Desirably, one of R₁, R₂ and R₃ is hydrogen, and the others arefluorine.

The compound of formula I may be provided as the (R) or (S) enantiomer,or as a mixture of the (R) and (S) enantiomers in any proportions,including the racemate. The compound of formula IA and of formula IBmost preferably consists of the (R)-enantiomer.

The compound may suitably be provided in the form of the hydrochloridesalt. However, given the secondary aliphatic amino group, it will beobvious to the skilled technician that other acid salts can be made andare within the scope of the claimed invention.

In an embodiment, the compound of formula IB has the formula B:

Thus, the present invention provides a compound of formula B for use intreating pulmonary arterial hypertension (PAH), wherein the compound offormula B includes its (R) or (S) enantiomer, a mixture of its (R) and(S) enantiomer, and pharmaceutically acceptable salts thereof.

The compound of formula B may be provided as the (R) or (S) enantiomer,or as a mixture of the (R) and (S) enantiomers in any proportions,including the racemate. Preferably the compound of formula B is providedas the R-enantiomer, (R)-B (compound B):

The compound of formula B (or (R)-B) is suitably provided as thehydrochloride salt. However, given the secondary aliphatic amino group,it will be obvious to the skilled technician that other acid salts canbe made and are within the scope of the claimed invention.

It will be appreciated that the term “compounds of formula I” as usedherein encompasses the compounds of formula I, IA, IB, B and (R)-B, andcompound A.

Particular compounds of formula I for use in treating pulmonary arterialhypertension include:

-   (S)-5-(2-aminoethyl)-1-(1,2,3,4-tetrahydronaphthalen-2-yl)-1,3-dihydroimidazole-2-thione;-   (S)-5-(2-aminoethyl)-1-(5,7-difluoro-1,2,3,4-tetrahydronaphthalen-2-yl)-1,3-dihydroimidazole-2-thione;-   (R)-5-(2-aminoethyl)-1-chroman-3-yl-1,3-dihydroimidazole-2-thione;-   (R)-5-(2-aminoethyl)-1-(6-hydroxychroman-3-yl)-1,3-dihydroimidazole-2-thione;-   (R)-5-(2-aminoethyl)-1-(8-hydroxychroman-3-yl)-1,3-dihydroimidazole-2-thione;-   (R)-5-(2-aminoethyl)-1-(6-methoxychroman-3-yl)-1,3-dihydroimidazole-2-thione;-   (R)-5-(2-aminoethyl)-1-(8-methoxychroman-3-yl)-1,3-dihydroimidazole-2-thione;-   (R)-5-(2-aminoethyl)-1-(6-fluorochroman-3-yl)-1,3-dihydroimidazole-2-thione;-   (R)-5-(2-aminoethyl)-1-(8-fluorochroman-3-yl)-1,3-dihydroimidazole-2-thione;-   (R)-5-(2-aminoethyl)-1-(6,7-difluorochroman-3-yl)-1,3-dihydroimidazole-2-thione;-   (R)-5-(2-aminoethyl)-1-(6,8-difluorochroman-3-yl)-1,3-dihydroimidazole-2-thione;-   (S)-5-(2-aminoethyl)-1-(6,8-difluorochroman-3-yl)-1,3-dihydroimidazole-2-thione;-   (R)-5-(2-aminoethyl)-1-(6,7,8-trifluorochroman-3-yl)-1,3-dihydroimidazole-2-thione;-   (R)-5-(2-aminoethyl)-1-(6-chloro-8-methoxychroman-3-yl)-1,3-dihydroimidazole-2-thione;-   (R)-5-(2-aminoethyl)-1-(6-methoxy-8-chlorochroman-3-yl)-1,3-dihydroimidazole-2-thione;-   (R)-5-(2-aminoethyl)-1-(6-nitrochroman-3-yl)-1,3-dihydroimidazole-2-thione;-   (R)-5-(2-aminoethyl)-1-(8-nitrochroman-3-yl)-1,3-dihydroimidazole-2-thione;-   (R)-5-(2-aminoethyl)-1-[6-(acetylamino)chroman-3-yl]-1,3-dihydroimidazole-2-thione;-   (R)-5-aminomethyl-1-chroman-3-yl-1,3-dihydroimidazole-2-thione;-   (R)-5-aminomethyl-1-(6-hydroxychroman-3-yl)-1,3-dihydroimidazole-2-thione;-   (R)-5-(2-aminoethyl)-1-(6-hydroxy-7-benzylchroman-3-yl)-1,3-dihydroimidazole-2-thione;-   (R)-5-aminomethyl-1-(6,8-difluorochroman-3-yl)-1,3-dihydroimidazole-2-thione;-   (R)-5-(3-aminopropyl)-1-(6,8-difluorochroman-3-yl)-1,3-dihydroimidazole-2-thione;-   (S)-5-(3-aminopropyl)-1-(5,7-difluoro-1,2,3,4-tetrahydronaphthalen-2-yl)-1,3-dihydroimidazole-2-thione;-   (R,S)-5-(2-aminoethyl)-1-(6-hydroxythiochroman-3-yl)-1,3-dihydroimidazole-2-thione;-   (R,S)-5-(2-aminoethyl)-1-(6-methoxythiochroman-3-yl)-1,3-dihydroimidazole-2-thione;-   (R)-5-(2-benzylaminoethyl)-1-(6-methoxychroman-3-yl)-1,3-dihydroimidazole7-2-thione;-   (R)-5-(2-benzylaminoethyl)-1-(6-hydroxychroman-3-yl)-1,3-dihydroimidazole-2-thione;-   (R)-1-(6-hydroxychroman-3-yl)-5-(2-methylaminoethyl)-1,3-dihydroimidazole-2-thione;-   (R)-1-(6,8-difluorochroman-3-yl)-5-(2-methylaminoethyl)-1,3-dihydroimidazole-2-thione;-   (R)-1-chroman-3-yl-5-(2-methylaminoethyl)-1,3-dihydroimidazole-2-thione;    or-   (R)-5-(2-(benzylamino)ethyl)-1-(6,8-difluorochroman-3-yl)-1H-imidazole-2(3H)-thione.

The compounds above may suitably be provided in the form of apharmaceutically acceptable salt, preferably the hydrochloride salt.

In an embodiment, there is provided(R)-5-(2-aminoethyl)-1-(6,8-difluorochroman-3-yl)-1,3-dihydroimidazole-2-thioneor(R)-5-(2-aminoethyl)-1-(6,8-difluorochroman-3-yl)-1,3-dihydroimidazole-2-thione,or a pharmaceutically acceptable salt thereof, typically thehydrochloride salt, for use in treating pulmonary arterial hypertension.Preferably, there is provided the hydrochloride salt of(R)-5-(2-aminoethyl)-1-(6,8-difluorochroman-3-yl)-1,3-dihydroimidazole-2-thioneor(R)-5-(2-aminoethyl)-1-(6,8-difluorochroman-3-yl)-1,3-dihydroimidazole-2-thionefor use in treating pulmonary arterial hypertension. The(R)-5-(2-aminoethyl)-1-(6,8-difluorochroman-3-yl)-1,3-dihydroimidazole-2-thioneor(R)-5-(2-aminoethyl)-1-(6,8-difluorochroman-3-yl)-1,3-dihydroimidazole-2-thione,or pharmaceutically acceptable salt thereof may be used in combinationwith one or more active pharmaceutical ingredients, as described below.

In an embodiment, there is provided(R)-5-(2-(benzylamino)ethyl)-1-(6,8-difluorochroman-3-yl)-1H-imidazole-2(3H)-thioneor(R)-5-(2-aminoethyl)-1-(6,8-difluorochroman-3-yl)-1,3-dihydroimidazole-2-thione,or pharmaceutically acceptable salt thereof, typically the hydrochloridesalt, for use in treating pulmonary arterial hypertension. Preferably,there is provided(R)-5-(2-(benzylamino)ethyl)-1-(6,8-difluorochroman-3-yl)-1H-imidazole-2(3H)-thioneor(R)-5-(2-aminoethyl)-1-(6,8-difluorochroman-3-yl)-1,3-dihydroimidazole-2-thione(hydrochloride) for use in treating pulmonary arterial hypertension. The(R)-5-(2-(benzylamino)ethyl)-1-(6,8-difluorochroman-3-yl)-1H-imidazole-2(3H)-thioneor(R)-5-(2-aminoethyl)-1-(6,8-difluorochroman-3-yl)-1,3-dihydroimidazole-2-thione,or pharmaceutically acceptable salt thereof may be used in combinationwith one or more active pharmaceutical ingredients, as described below.

The preparation of compounds of formula I is described in WO2004/033447and WO2008/136695.

Reference is made to the “Guidelines for the diagnosis and treatment ofpulmonary hypertension” (European Heart Journal (2009) 30, 2493-2537)for details on the definition, classification and pathology andpathobiological features of PAH.

Typically, pulmonary hypertension is a group of diseases characterizedby a progressive increase of pulmonary vascular resistance leading toright ventricular failure and premature death. It may be defined by amean pulmonary artery pressure greater than 25 mmHg at rest.

PAH has been clinically classified by the WHO into 5 groups, accordingto the cause of the disease, and symptoms may differ, depending on the‘group’ that caused the disease. However, ‘common’ symptoms are asfollows:

Difficulty in breathing or shortness of breath (main symptom)

Fatigue

Dizziness

Swelling in the ankles or legs (edema)

Bluish lips and skin (cyanosis)

Chest pain

Racing pulse and palpitations

A clinical classification of PAH has been undertaken and reported bySimonneau G et al in “Updated clinical classification of pulmonaryhypertension”, J Am Coll Cardiol 2009; 54:S43-S54. PAH was classified asfollows:

Clinical Classification of Pulmonary Arterial Hypertension (PAH): 1.1Idiopathic 1.2 Heritable

1.2.1 Bone morphogenetic protein receptor type II (BMPR2)

1.2.2 Activin receptor-like kinase-1 (ALK1), endoglin (with or withouthereditary haemorrhagic telangiectasia)

1.2.3 Unknown

1.3 Drugs and toxins induced1.4 Associated with (Associated PAH, APAH)

1.4.1 Connective tissue diseases

1.4.2 HIV infection

1.4.3 Portal hypertension

1.4.4 Congenital heart disease

1.4.5 Schistosomiasis

1.4.6 Chronic haemolytic anaemia

1.5 Persistent pulmonary hypertension of the newborn

The WHO has also provided the following functional assessmentclassification:

Functional Class Symptomatic Profile

-   I Patients with pulmonary hypertension but without resulting    limitation of physical activity. Ordinary physical activity does not    cause dyspnoea or fatigue, chest pain, or near syncope-   II Patients with pulmonary hypertension resulting in slight    limitation of physical activity. They are comfortable at rest.    Ordinary physical activity causes undue dyspnoea or fatigue, chest    pain, or near syncope-   III Patients with pulmonary hypertension resulting in marked    limitation of physical activity. They are comfortable at rest. Less    than ordinary activity causes undue dyspnoea or fatigue, chest pain,    or near syncope-   IV Patients with pulmonary hypertension with inability to carry out    any physical activity without symptoms. These patients manifest    signs of right heart failure. Dyspnoea and/or fatigue may even be    present at rest. Discomfort is increased by any physical activity

In an embodiment, the present invention relates to a compound of formulaI in particular,(R)-5-(2-(benzylamino)ethyl)-1-(6,8-difluorochroman-3-yl)-1H-imidazole-2(3H)-thioneor(R)-5-(2-aminoethyl)-1-(6,8-difluorochroman-3-yl)-1,3-dihydroimidazole-2-thione(hydrochloride), whether alone or in combination with one or more otheractive pharmaceutical ingredients (APIs), for use in treating one ormore of the above classes of PAH. The PAH may be characterised by a meanpulmonary artery pressure greater than 25 mmHg at rest.

In an embodiment, the present invention relates to a compound of formulaI in particular,(R)-5-(2-(benzylamino)ethyl)-1-(6,8-difluorochroman-3-yl)-1H-imidazole-2(3H)-thioneor(R)-5-(2-aminoethyl)-1-(6,8-difluorochroman-3-yl)-1,3-dihydroimidazole-2-thione(hydrochloride), whether alone or in combination with one or more otheractive pharmaceutical ingredients, for use in treating one or more ofthe above classes of PAH. The PAH may be characterised by a meanpulmonary artery pressure greater than 25 mmHg at rest.

As used herein, the term treatment and variations such as ‘treat’ or‘treating’ refer to any regime that can benefit a human or non-humananimal. The treatment may be in respect of an existing condition or maybe prophylactic (preventative treatment). Treatment may includecurative, alleviation or prophylactic effects. The treatment may alsoinvolve curing, alleviating or preventing symptoms associated with thedisorder rather than acting on the underlying cause of the disorder.Treatment with a compound of formula I in combination with one of theother classes of compounds includes simultaneous and sequentialadministration of the two or more drugs.

The expected beneficial therapeutic effects of the compounds of formulaI in particular,(R)-5-(2-(benzylamino)ethyl)-1-(6,8-difluorochroman-3-yl)-1H-imidazole-2(3H)-thioneor(R)-5-(2-aminoethyl)-1-(6,8-difluorochroman-3-yl)-1,3-dihydroimidazole-2-thione(hydrochloride) in treating pulmonary arterial hypertension have beendescribed above. It will be appreciated that the compounds of formula Iin particular,(R)-5-(2-(benzylamino)ethyl)-1-(6,8-difluorochroman-3-yl)-1H-imidazole-2(3H)-thioneor(R)-5-(2-aminoethyl)-1-(6,8-difluorochroman-3-yl)-1,3-dihydroimidazole-2-thione(hydrochloride) may also exhibit beneficial therapeutic effects intreating conditions associated with PAH, for example conditionsexhibiting lung injury. Such conditions include breathing difficulties,shortness of breath, fatigue, dizziness, swelling in the ankles or legs(oedema), bluish lips and skin (cyanosis), chest pain, racing pulse andpalpitations Treatment of such conditions with compounds of formula I,in particular,(R)-5-(2-(benzylamino)ethyl)-1-(6,8-difluorochroman-3-yl)-1H-imidazole-2(3H)-thioneor(R)-5-(2-aminoethyl)-1-(6,8-difluorochroman-3-yl)-1,3-dihydroimidazole-2-thione,or pharmaceutically acceptable salts thereof forms another aspect of thepresent invention. Thus, the present invention provides a compound offormula I in particular,(R)-5-(2-(benzylamino)ethyl)-1-(6,8-difluorochroman-3-yl)-1H-imidazole-2(3H)-thioneor(R)-5-(2-aminoethyl)-1-(6,8-difluorochroman-3-yl)-1,3-dihydroimidazole-2-thione,or a pharmaceutically acceptable salt thereof for use in treating lunginjury. Suitably, the present invention provides a compound of formula Iin particular,(R)-5-(2-(benzylamino)ethyl)-1-(6,8-difluorochroman-3-yl)-1H-imidazole-2(3H)-thioneor(R)-5-(2-aminoethyl)-1-(6,8-difluorochroman-3-yl)-1,3-dihydroimidazole-2-thione,or a pharmaceutically acceptable salt thereof for use in treating one ormore of the following conditions: breathing difficulties, shortness ofbreath, fatigue, dizziness, swelling in the ankles or legs (oedema),bluish lips and skin (cyanosis), chest pain, racing pulse andpalpitations.

Clinical trials may be undertaken in order to demonstrate thetherapeutic efficacy of a compound of formula I, in particular,(R)-5-(2-(benzylamino)ethyl)-1-(6,8-difluorochroman-3-yl)-1H-imidazole-2(3H)-thioneor(R)-5-(2-aminoethyl)-1-(6,8-difluorochroman-3-yl)-1,3-dihydroimidazole-2-thione(hydrochloride), whether alone or in combination with one or more otheractive pharmaceutical ingredients, in treating PAH. Preferably, theclinical trial is a randomised controlled trial; suitably the trialbeing a double-blind trial.

According to another aspect of the present invention, there is provideda method of treating pulmonary arterial hypertension comprisingadministering a therapeutically effective amount of a compound offormula I, in particular,(R)-5-(2-(benzylamino)ethyl)-1-(6,8-difluorochroman-3-yl)-1H-imidazole-2(3H)-thioneor(R)-5-(2-aminoethyl)-1-(6,8-difluorochroman-3-yl)-1,3-dihydroimidazole-2-thione,or a pharmaceutically acceptable salt thereof as described above to asubject in need thereof, optionally in combination with another API suchas one listed below. The PAH may be characterised by a mean pulmonaryartery pressure greater than 25 mmHg at rest.

According to another aspect of the present invention, there is provideda method of treating lung injury comprising administering atherapeutically effective amount of a compound of formula I, inparticular,(R)-5-(2-(benzylamino)ethyl)-1-(6,8-difluorochroman-3-yl)-1H-imidazole-2(3H)-thioneor(R)-5-(2-aminoethyl)-1-(6,8-difluorochroman-3-yl)-1,3-dihydroimidazole-2-thione,or a pharmaceutically acceptable salt thereof as described above to asubject in need thereof, optionally in combination with another API suchas one listed below. Suitably, the present invention provides a methodof treating one or more of the following conditions: breathingdifficulties, shortness of breath, fatigue, dizziness, swelling in theankles or legs (oedema), bluish lips and skin (cyanosis), chest pain,racing pulse and palpitations comprising administering a therapeuticallyeffective amount of a compound of formula I, in particular,(R)-5-(2-(benzylamino)ethyl)-1-(6,8-difluorochroman-3-yl)-1H-imidazole-2(3H)-thioneor(R)-5-(2-aminoethyl)-1-(6,8-difluorochroman-3-yl)-1,3-dihydroimidazole-2-thione,or a pharmaceutically acceptable salt thereof as described above to asubject in need thereof, optionally in combination with another API suchas one listed below.

According to another aspect of the present invention, there is providedthe use of a compound of formula I, in particular,(R)-5-(2-(benzylamino)ethyl)-1-(6,8-difluorochroman-3-yl)-1H-imidazole-2(3H)-thioneor(R)-5-(2-aminoethyl)-1-(6,8-difluorochroman-3-yl)-1,3-dihydroimidazole-2-thione,or a pharmaceutically acceptable salt thereof as described above,optionally in combination with another API such as one listed below, inthe manufacture of a medicament for treating pulmonary arterialhypertension. The PAH may be characterised by a mean pulmonary arterypressure greater than 25 mmHg at rest.

According to another aspect of the present invention, there is providedthe use of a compound of formula I, in particular,(R)-5-(2-(benzylamino)ethyl)-1-(6,8-difluorochroman-3-yl)-1H-imidazole-2(3H)-thioneor(R)-5-(2-aminoethyl)-1-(6,8-difluorochroman-3-yl)-1,3-dihydroimidazole-2-thione,or a pharmaceutically acceptable salt thereof as described above,optionally in combination with another API such as one listed below, inthe manufacture of a medicament for treating lung injury. Suitably, thepresent invention provides the use of a compound of formula I, inparticular,(R)-5-(2-(benzylamino)ethyl)-1-(6,8-difluorochroman-3-yl)-1H-imidazole-2(3H)-thioneor(R)-5-(2-aminoethyl)-1-(6,8-difluorochroman-3-yl)-1,3-dihydroimidazole-2-thione,or a pharmaceutically acceptable salt thereof as described above,optionally in combination with another API such as one listed below, inthe manufacture of a medicament for treating one or more of thefollowing: breathing difficulties, shortness of breath, fatigue,dizziness, swelling in the ankles or legs (oedema), bluish lips and skin(cyanosis), chest pain, racing pulse and palpitations.

The compound of formula I, in particular,(R)-5-(2-(benzylamino)ethyl)-1-(6,8-difluorochroman-3-yl)-1H-imidazole-2(3H)-thioneor(R)-5-(2-aminoethyl)-1-(6,8-difluorochroman-3-yl)-1,3-dihydroimidazole-2-thione,or a pharmaceutically acceptable salt thereof, suitably for use asdescribed above, may be formulated into a pharmaceutical composition,optionally in combination with another API such as one listed below.

For the preparation of pharmaceutical compositions of compounds offormula I, in particular,(R)-5-(2-(benzylamino)ethyl)-1-(6,8-difluorochroman-3-yl)-1H-imidazole-2(3H)-thioneor(R)-5-(2-aminoethyl)-1-(6,8-difluorochroman-3-yl)-1,3-dihydroimidazole-2-thione(hydrochloride), inert pharmaceutically acceptable carriers are admixedwith the active compounds. The pharmaceutically acceptable carriers maybe either solid or liquid. Solid form preparations include powders,tablets, dispersible granules and capsules. A solid carrier can be oneor more substances which may also act as diluents, flavouring agents,solubilizers, lubricants, suspending agents, binders or tabletdisintegrating agents; it may also be an encapsulating material.

Preferably the pharmaceutical composition is in unit dosage form, e.g.packaged composition, the package containing discrete quantities of thecomposition such as packeted tablets, capsules and powders in vials orampoules. The dosages may be varied depending on the requirement of thepatient, the severity of the disease and the particular compound beingemployed. For convenience, the total daily dosage may be divided andadministered in portions throughout the day. It is expected that once ortwice per day administration will be most suitable.

When the compound of formula I is in the form of Compound B((R)-5-(2-(benzylamino)ethyl)-1-(6,8-difluorochroman-3-yl)-1H-imidazole-2(3H)-thione),the dose of Compound B is in the range of about 10 mg/day to about 50mg/day, preferably in the range of about 15 mg/day to about 45 mg/day,more preferably in the range of about 20 mg/day to about 40 mg/day, mostpreferably in the range of about 25 mg/day to about 35 mg/day typicallyabout 30 mg/day. Preferably, the Compound B is in the form of a singledaily dosage. The present invention thus also provides a pharmaceuticalcomposition comprising Compound B in the amount described above for usein treating PAH. The PAH may be characterised by a mean pulmonary arterypressure greater than 25 mmHg at rest.

The composition may further comprise another active pharmaceuticalingredient. Suitable active ingredients are described in WO2007/081232and WO2008/136695.

The compound of formula I, in particular,(R)-5-(2-(benzylamino)ethyl)-1-(6,8-difluorochroman-3-yl)-1H-imidazole-2(3H)-thioneor(R)-5-(2-aminoethyl)-1-(6,8-difluorochroman-3-yl)-1,3-dihydroimidazole-2-thione(hydrochloride), may be used in conjunction with another activepharmaceutical ingredient, i.e. with one or more other classes ofcompounds. The compound of formula I, in particular,(R)-5-(2-(benzylamino)ethyl)-1-(6,8-difluorochroman-3-yl)-1H-imidazole-2(3H)-thioneor(R)-5-(2-aminoethyl)-1-(6,8-difluorochroman-3-yl)-1,3-dihydroimidazole-2-thione(hydrochloride), may be used in simultaneous or sequentialadministration. For simultaneous administration the compound of formulaI, in particular,(R)-5-(2-(benzylamino)ethyl)-1-(6,8-difluorochroman-3-yl)-1H-imidazole-2(3H)-thioneor(R)-5-(2-aminoethyl)-1-(6,8-difluorochroman-3-yl)-1,3-dihydroimidazole-2-thione(hydrochloride), may be incorporated into the same composition as afixed dose composition, as noted above, or may be administered as twoseparate compositions. Suitable active ingredients are described inWO2007/081232 and WO2008/136695.

Typically, the other active ingredient is selected from the followingone or more of the following classes:

diuretics; beta-adrenergic antagonists; alpha2-adrenergic agonists;alpha1-adrenergic antagonists; dual beta- and alpha-adrenergicantagonists; calcium channel blockers; potassium channel activators;anti-arrhythmics; ACE inhibitors; angiotensin (e.g. AT1) receptorantagonists; renin inhibitors; lipid lowerers, vasopeptidase inhibitors;nitrates; endothelin antagonists/endothelin receptor inhibitors; neutralendopeptidase inhibitors; anti-angiotensin vaccines; vasodilators;phosphodiesterase inhibitors; cardiac glycosides; serotonin antagonists;CNS acting agents; calcium sensitisers; HMG CoA reductase inhibitors;vasopressin antagonists; adenosine Al receptor antagonists; atrialnatriuretic peptide (ANP) agonists; chelating agents;corticotrophin-releasing factor receptor; glucagon-like peptide-1agonists; sodium, potassium ATPase inhibitors; advanced glycosylationend-products (AGE) crosslink breakers; mixedneprilysin/endothelin-converting enzyme (NEP/ECE) inhibitors; nociceptinreceptor (ORL-1) agonists (e.g. alprazolam); xanthine oxidaseinhibitors; benzodiazepine agonists; cardiac myosin activators; chymaseinhibitors; endothelial nitric oxide synthase (ENOS) transcriptionenhancers; and neutral endopeptidase inhibitors such as thiorphan, inparticular angiotensin receptot antagonists, phosphodiesteraseinhibitors and endothelin receptor inhibitors.

The invention also envisages the use of nepicastat with the classes ofcompounds described above.

The other active ingredient may be selected from one or more of thefollowing: epoprostenol (for example continuously injected through anintravenous (IV) catheter), iloprost (for example inhaled), bosentan,ambrisentan, sitaxentan, sildenafil, tadalafil, amlodipine, felodipine,diltiazem, nifedipine, nicardipine isosorbide dinitrate,isosorbide-5-mononitrate, warfarin, captopril, enalapril, lisinopril,benazepril, fosinopril, trandolapril, quinapril, ramipril, perindopril,zofenopril, cilazapril, imidapril, losartan, candersartan, olmesartan,irbesartan, eprosartan, telmisartan, valsartan, acetazolamide,dichlorphenamide, methazolamide, furosemide, ethacrynic acid, torasemide(torsemide), azosemide (axosemide), piretanide, tripamide,hydrochlorothiazide, chlorothiazide, bendroflumethiazide,methyclothiazide, polythiazide, trichlormethiazide, chlorthalidone,indapamide, metolazone, quinethazone, amiloride, triamterene,spironolactone, canrenone, potassium canrenoate and eplerenone.Suitably, one of the above other active ingredients is combined with thecompound of formula I, in particular,(R)-5-(2-(benzylamino)ethyl)-1-(6,8-difluorochroman-3-yl)-1H-imidazole-2(3H)-thioneor(R)-5-(2-aminoethyl)-1-(6,8-difluorochroman-3-yl)-1,3-dihydroimidazole-2-thione(hydrochloride).

Other APIs used in treating pulmonary arterial hypertension may also becombined with the compound of formula I.

In an embodiment, the compound of formula I, in particular,(R)-5-(2-(benzylamino)ethyl)-1-(6,8-difluorochroman-3-yl)-1H-imidazole-2(3H)-thioneor(R)-5-(2-aminoethyl)-1-(6,8-difluorochroman-3-yl)-1,3-dihydroimidazole-2-thione(hydrochloride), is used in combination with an endothelin antagonist.Suitably, the endothelin antagonist is bosentan, sitaxentan orambrisentan.

In an embodiment, the compound of formula I, in particular,(R)-5-(2-(benzylamino)ethyl)-1-(6,8-difluorochroman-3-yl)-1H-imidazole-2(3H)-thioneor(R)-5-(2-aminoethyl)-1-(6,8-difluorochroman-3-yl)-1,3-dihydroimidazole-2-thione(hydrochloride), is used in combination with a prostacyclin analogue.Suitably, the prostacyclin analogue is selected from epoprostenol (forexample continuously injected through an intravenous (IV) catheter),iloprost (for example inhaled), treprostinil and beraprost.

In an embodiment, the compound of formula I, in particular,(R)-5-(2-(benzylamino)ethyl)-1-(6,8-difluorochroman-3-yl)-1H-imidazole-2(3H)-thioneor(R)-5-(2-aminoethyl)-1-(6,8-difluorochroman-3-yl)-1,3-dihydroimidazole-2-thione(hydrochloride), is used in combination with a phosphodiesterase type-5(PDE-5) inhibitor. Suitably, the PDE-5 inhibitor is selected fromsildenafil and tadalafil.

In an embodiment, the compound of formula I, in particular,(R)-5-(2-(benzylamino)ethyl)-1-(6,8-difluorochroman-3-yl)-1H-imidazole-2(3H)-thioneor(R)-5-(2-aminoethyl)-1-(6,8-difluorochroman-3-yl)-1,3-dihydroimidazole-2-thione(hydrochloride), is used in combination with a calcium channel blocker(CCB). Suitably, the CCB is selected from nifedipine, dilitiazem,amlodipine and felodipine.

According to another aspect of the present invention, there is provideda pharmaceutical composition comprising a compound of formula I, inparticular,(R)-5-(2-(benzylamino)ethyl)-1-(6,8-difluorochroman-3-yl)-1H-imidazole-2(3H)-thioneor(R)-5-(2-aminoethyl)-1-(6,8-difluorochroman-3-yl)-1,3-dihydroimidazole-2-thione,or a pharmaceutically acceptable salt thereof as described above and anendothelin antagonist. Suitably, the endothelin antagonist is bosentan.The pharmaceutical combination may also include one or more inertpharmaceutically acceptable carriers.

In an embodiment, there is provided a pharmaceutical combinationcomprising a compound of formula IA or a pharmaceutically acceptablesalt thereof as described above and an endothelin antagonist.Preferably, the combination comprises(R)-5-(2-aminoethyl)-1-(6,8-difluorochroman-3-yl)-1,3-dihydroimidazole-2-thionehydrochloride and an endothelin antagonist. More preferably, thecombination comprises(R)-5-(2-aminoethyl)-1-(6,8-difluorochroman-3-yl)-1,3-dihydroimidazole-2-thionehydrochloride and bosentan. The pharmaceutical combination may alsoinclude one or more inert pharmaceutically acceptable carriers.

In an embodiment, there is provided a pharmaceutical combinationcomprising a compound of formula IB or a pharmaceutically acceptablesalt thereof as described above and an endothelin antagonist.Preferably, the compound of formula IB is a compound of formula (R)-B.More preferably, the combination comprises(R)-5-(2-(benzylamino)ethyl)-1-(6,8-difluorochroman-3-yl)-1H-imidazole-2(3H)-thioneand an endothelin antagonist. Most preferably, the combination comprises(R)-5-(2-(benzylamino)ethyl)-1-(6,8-difluorochroman-3-yl)-1H-imidazole-2(3H)-thioneand bosentan. The pharmaceutical combination may also include one ormore inert pharmaceutically acceptable carriers.

Preferably the pharmaceutical composition comprising the combination ofthe compound of formula I and the other active pharmaceutical ingredientis in unit dosage form, e.g. packaged composition, the packagecontaining discrete quantities of the composition such as packetedtablets, capsules and powders in vials or ampoules. Preferably, thecompound of formula I in combination with the other activepharmaceutical ingredient is in the form of a single daily dosage, i.e.a unit dosage form to be administered once a day.

When the compound of formula IB is in the form of Compound B((R)-5-(2-(benzylamino)ethyl)-1-(6,8-difluorochroman-3-yl)-1H-imidazole-2(3H)-thione),the dose of Compound B may be in the range of about 10 mg/day to about50 mg/day, preferably in the range of about 15 mg/day to about 45mg/day, more preferably in the range of about 20 mg/day to about 40mg/day, most preferably in the range of about 25 mg/day to about 35mg/day typically about 30 mg/day. When the Compound B is combined withbosentan, the bosentan may be in the range of about 50 mg/day to about350 mg/day, preferably in the range of about 75 mg/day to about 325mg/day.

The dose of bosentan in the pharmaceutical composition comprising acombination of Compound B and bosentan may be in the in the range ofabout 80 mg/day to about 120 mg/day, most preferably in the range ofabout 90 mg/day to about 110 mg/day typically about 100 mg/day. Thus, inone embodiment, the dosages of Compound B and bosentan may be: CompoundB in the range of about 10 mg/day to about 50 mg/day; and bosentan inthe range of about 80 mg/day to about 120 mg/day.

The dose of bosentan in the pharmaceutical composition comprising acombination of Compound B and bosentan may be in the in the range ofabout 280 mg/day to about 320 mg/day, most preferably in the range ofabout 290 mg/day to about 310 mg/day typically about 300 mg/day. Thus,in one embodiment, the dosages of Compound B and bosentan may be:Compound B in the range of about 10 mg/day to about 50 mg/day; andbosentan in the range of about 280 mg/day to about 320 mg/day.

Preferably, the Compound B and bosentan is in the form of a single dailydosage. The present invention thus also provides a pharmaceuticalcomposition comprising Compound B and bosentan in the amounts describedabove for use in treating PAH. The PAH may be characterised by a meanpulmonary artery pressure greater than 25 mmHg at rest.

According to another aspect of the present invention, there is provideda pharmaceutical composition comprising a compound of formula I, inparticular,(R)-5-(2-(benzylamino)ethyl)-1-(6,8-difluorochroman-3-yl)-1H-imidazole-2(3H)-thioneor(R)-5-(2-aminoethyl)-1-(6,8-difluorochroman-3-yl)-1,3-dihydroimidazole-2-thione,or a pharmaceutically acceptable salt thereof as described above andphosphodiesterase type-5 (PDE-5) inhibitor. Suitably, thephosphodiesterase type-5 (PDE-5) inhibitor is sildenafil. Thepharmaceutical combination may also include one or more inertpharmaceutically acceptable carriers.

In an embodiment, there is provided a pharmaceutical combinationcomprising a compound of formula IA or a pharmaceutically acceptablesalt thereof as described above and an endothelin antagonist.Preferably, the combination comprises(R)-5-(2-aminoethyl)-1-(6,8-difluorochroman-3-yl)-1,3-dihydroimidazole-2-thionehydrochloride and phosphodiesterase type-5 (PDE-5) inhibitor. Morepreferably, the combination comprises(R)-5-(2-aminoethyl)-1-(6,8-difluorochroman-3-yl)-1,3-dihydroimidazole-2-thionehydrochloride and sildenafil. The pharmaceutical combination may alsoinclude one or more inert pharmaceutically acceptable carriers.

In an embodiment, there is provided a pharmaceutical combinationcomprising a compound of formula IB or a pharmaceutically acceptablesalt thereof as described above and an endothelin antagonist.Preferably, the compound of formula IB is a compound of formula (R)-B.More preferably, the combination comprises(R)-5-(2-(benzylamino)ethyl)-1-(6,8-difluorochroman-3-yl)-1H-imidazole-2(3H)-thioneand a phosphodiesterase type-5 (PDE-5) inhibitor. Most preferably, thecombination comprises(R)-5-(2-(benzylamino)ethyl)-1-(6,8-difluorochroman-3-yl)-1H-imidazole-2(3H)-thioneand sildenafil. The pharmaceutical combination may also include one ormore inert pharmaceutically acceptable carriers.

BRIEF DESCRIPTION OF THE DRAWINGS

Reference is now made to the accompanying Figures, in which:

FIG. 1—represents a schematic of the study described below.

FIG. 2—Kaplan-Meyer survival curves from the day of MCT administrationfrom an initial n=20 of rats in each group of animals.

FIG. 3—Kaplan-Meyer survival curves from the day of MCT administrationfrom an initial n=20 of rats in each group of animals, except theMCT+Bos300 (n=10).

FIG. 4—Ratios of heart weight over bodyweight (BW) (FIG. 4a ) and Fultonindex (RV/LF+S) (FIG. 4b ) from MCT (●), MCT+Bos300 (▪), MCT+Bos100 (▴),MCT+Compound B (▾), MCT+Compound B+Bos100 (♦), SHAM (∘) andSHAM+Compound B (□) treated rats at 28 days after MCT or vehicle (Sham)administration. Data represents means±sem of n=4-14. Significantlydifferent from MCT (*P<0.05) or from Sham and Sham+Compound B (#P<0.05).

FIG. 5—Ratios of right ventricle (RV) (FIG. 5a ) and left ventricle plusseptum (LV/S) weight over bodyweight (BW) (FIG. 5b ) from MCT (●),MCT+Bos300 (▪), MCT+Bos100 (▴), MCT+Compound B (▾), MCT+CompoundB+Bos100 (♦), SHAM (∘) and SHAM+Compound B (□) treated rats at 28 daysafter MCT or vehicle (Sham) administration. Data represents means±sem ofn=4-14. Significantly different from MCT (*P<0.05) or from Sham andSham+Compound B (# P<0.05).

MATERIALS AND METHODS In Vivo Studies

One of the most-used animal models is the Monocrotaline (MCT) lunginjury model of Pulmonary Arterial Hypertension. Administration of MCT,that is metabolized in the liver to the reactive metabolitedehydromonocrotaline, results in a syndrome characterized by acute lunginjury, interstitial pulmonary fibrosis, necrotizing pulmonaryarteritis, pulmonary hypertension, right ventricular (RV) hypertrophy,myocarditis and hepatic venoocclusive disease.

Experimental Procedure The Monocrotaline (MCT) Lung Injury Model ofPulmonary Arterial Hypertension Set Up

The protocol to use is a standard one (Curr. Protoc. Pharmacol.46:5.56.1-5.56.11(2009) John Wiley & Sons Inc). Rats, of 6-7 weeks old(150-200 g) are administered MCT 60 mg/kg subcutaneously, and thepathology usually develops in the course of initial 3 to 4 weeks withthe animals dying within the next 2 weeks (5^(th)-6^(th) week). Adecrease of 50% in food and water consumption is thought to beindicative of pathological status severe enough for testing.

Experimental Design

Two studies were conducted separately and a combination analysisperformed on the two studies. Each study consisted in the schemerepresented in FIG. 1.

Compound B refers to(R)-5-(2-benzylamino)ethyl)-1-(6,8-difluorochroman-3-yl)-1H-imidazole-2(3H)-thione(i.e. compound (R)-B as shown above). The structure is as follows:

The treatment groups for each study were as follows.

Group Treatment n G1 MCT + Vehicle 10 G2 MCT + Bosentan 300 mg/kg 10 G3MCT + Bosentan 100 mg/kg 10 G4 MCT + Compound B 30 mg/kg 10 G5 MCT +Compound B 30 mg/kg + 10 Bosentan 100 mg/kg G6 Vehicle SHAM 6 G7Compound B 30 mg/kg SHAM 6

Compound B was obtained from Bial—Portela & Ca., S.A. Monocrotaline wasobtained from Sequoia Research Products Limited. Bosentan (CAS number157212-55-0) was obtained from APICHEM.

Abbreviations:

a. Bos=Bosentan

b. Bos100=Bosentan 100 mg/kg

c. Bos300=Bosentan 300 mg/kg

d. LV+S=Left ventricle+septum

e. MCT=Monocrotaline

f. RV=Right ventricle

The experimental design of both studies was the same, with animalsrandomized in seven groups, five groups of 10 animals each (MCT,MCT+Bos300, MCT+Bos100, MCT+Compound B, MCT+Compound B+Bos100) and 2groups of 6 animals each (SHAM and SHAM+Compound B).

The experimental details are described in more detail below; briefly,compounds were given to rats mixed with food starting, Compound B (30mg/kg/day) for groups MCT+Compound B and SHAM+Compound B, bosentan (300mg/kg/day) for group MCT+Bos300, bosentan (100 mg/kg/day) for groupMCT+Bos100, Compound B+bosentan (30+100 mg/kg/day) for groupMCT+Compound B+Bos100, and no compound for groups MCT and SHAM. Animalsfrom groups MCT were administered MCT and animals from groups SHAM wereadministered vehicle. Twenty eight days later, surviving animals weresacrificed, tissues collected and weighed. During the entireexperimental period food, water consumption and animal weight weremonitored. The ratios organ weight to body weight (BW) were calculatedto evaluate organ hypertrophy. The index of RV/LV+S was calculated as anindex of RV hypertrophy. In each study an n=10 animals was used for eachMCT treatment group, and an n=6 was used for the groups not given MCT.The animals from MCT+Bos300 group from Study 1 were not considered forthe combined survival analysis since these unexpectedly presented ahigher mortality effect as compared to the MCT group. The survivinganimals from this group were nevertheless considered for the cardiacremodelling analysis.

Administration volumes: 3 ml/kg for subcutaneous administration.

Storage conditions: The substances will be stored according toconditions defined in the respective certificate of analysis.

Data Acquisition and Analysis

Raw data acquisition was performed with Gilson UniPoint™ System Softwareversion 5.11. All data analysis was performed using Prism 5 for Windowssoftware, version 5.02 (GraphPad Software Inc., San Diego, Calif.).

Statistical Analysis

All data is expressed as mean±sem (Standard Error of the Mean) unlessotherwise indicated. Differences between treatment groups were analysedby one-way ANOVA and post-hoc Newman-Keuls multiple comparison test.P<0.05 was considered statistically significant.

Survival curves were derived by the product limit method of Kaplan andMeier, and compared using the logrank (Mantel Cox) test.

Study 1—Further Details

Animals were given powdered food for an adaptation period of 3-5 daysbefore treatment start. Compounds were given to rats mixed with food(powdered diet) starting on day −2. Forty-eight hours later, on day zeroanimals from groups GI through G5 were administered MCT subcutaneously(60 mg/kg) while remaining groups (G6 and G7) were given vehicle insteadof MCT. Treatment was prolonged for 28 days. Food, water consumption andanimal weight were monitored over the entire duration of the experiment.

On day 28 surviving animals were sacrificed and the followingtissues/organs collected and weighed: liver, kidney, lung, heart andspleen. Femurs were removed and measured. Heart was dissected into rightventricle, left ventricle+septum and these were also weighed. The ratioorgan to body weight (BW) was calculated as well as the ratio organweight to femur length. The index of RV/LV+S is an index of RVhypertrophy. Catecholamines were evaluated only on right ventricle andleft ventricle+septum.

Six weeks old male Wistar rats (151-187 g) were obtained from Harlan(Spain). Rats were kept 5 per cage, under controlled environmentalconditions (12 hr light/dark cycle and room temperature 22±1° C.).

All animal groups were fed powdered food, MCT (G1, n=10) and SHAM (G6,n=6) were given normal food and all other groups were given food mixedwith the compounds: MCT-Bos300 (G2, n=10) received food with 300mg/kg/day bosentan, MCT-Bos100 (G3, n=10) received food with 100mg/kg/day bosentan, MCT-1058 (G4, n=10) received food with 30 mg/kg/dayBIA 5-1058, MCT-1058-Bos100 (G5, n=10) received food with 100 mg/kg/daybosentan plus 30 mg/kg/day BIA 5-1058 and SHAM-1058 (G7, n=6) receivedfood with 30 mg/kg/day BIA 5-1058.

Forty eight hours later animals groups 1 through 5 were administered MCT(60 mg/kg) and groups 6 and 7 were administered vehicle subcutaneouslyin a volume of 3 ml/kg.

MCT was prepared by dissolving at 300 mg/kg in 1M HCl, neutralizing with1 M NaOH and diluting to 20 mg/ml with sterile water.

Water and food consumption for each cage and weights of the individualanimals were measured twice a week.

Animals were kept under treatment for 28 days after MCT administration.The animals that survived were, at that time, anaesthetized withpentobarbital 60 mg/kg administered intraperitoneally, and subjected totissue and organ collection.

Whole spleen, whole liver, left kidney and lungs were removed, cleanedand weighed. Heart was removed, cleaned from atria and vascular tissueand weighed. Right ventricle was then dissected from the rest of theheart (left ventricle+septum) and both were weighed and put in tubescontaining 0.2 M PCA. Femurs were removed, cleaned and length measuredwith a calliper.

The ratios organ weight to body weight and organ weight to femur lengthwere calculated as well as the ratio right ventricular weight to leftventricular+septum weight.

Tissues were left in 0.2 M PCA (perchloric acid) for 24 h in the dark at4° C. and were then frozen at −80° C. Catecholamines were measured inright ventricle and left ventricle+septum.

Catecholamines Quantification

Reagents and Materials: All reagents were obtained from Sigma-Aldrich.SPE columns Sep-Pak® Vac Alumina A cartridge 1 cc/100 mg 50-300 im100/box were obtained from Waters.

Tissues: Frozen tissues in 0.2 M PCA were thawed, the liquid phaseremoved, and filtered through 0.22 m Spin-X filters (Corning Costar) bycentrifugation in microfuge for 10 minutes at ˜5000 rpm, 4° C.Noradrenaline and dopamine were quantified in filtrates by high-pressureliquid chromatography with electrochemical detection (HPLC-ED).

Test system: Gilson HPLC-ED 142

Test Method: Chromatographic conditions were:

-   Flow rate: 1 ml/min-   Analytical Column: Spheri-5 RP18 5 μm, 4.6×250 mm, Perkin Elmer    serial #28918, lot#07H8-1318806-   Temperature: Ambient-   Injection volume: 50 μl-   Mobile phase: 0.15 mM EDTA, 0.1M Sodium acetate, 0.1M Citric acid    monohydrate, 1 mM Octyl sulphate, 1.0 mM Dibutylamine, 10% Methanol,    pH 3.5 with PCA-   Detector set at: Mode Amperometric    -   0.75 V potential    -   2 nA sensitivity

Study 2—Further Details

Animals were given powdered food for an adaptation period of 3-5 daysbefore treatment start. Compounds were given to rats mixed with food(powdered diet) starting on day −2. Forty-eight hours later, on day zeroanimals from groups GI through G5 were administered MCT subcutaneously(60 mg/kg) while remaining groups (G6 and G7) were given vehicle insteadof MCT. Animal weight, food and water consumption were evaluated twice aweek and animals were regularly observed for any disease signs.Mortality was registered and on day 28 surviving animals weresacrificed, selected organs weighed and femurs removed and measured.

Organs weighed were: liver, kidney, lung, heart and spleen. Heart wasdissected into right ventricle, left ventricle+septum and these werealso weighed. The ratio organ to body weight (BW) was calculated as wellas the ratio organ weight to femur length. The index of RV/LV+S is anindex of RV hypertrophy.

Rats were housed in groups of 5 in macrolon cages on wood litter withfree access to powder chow diet (Code 113-SAFE, 89290 Augy, France) andtap water. The animal house was maintained in a 12-hour light/dark cycle(0700 to 1900 hours) in a controlled ambient temperature of 22±1° C.

Treatment with compound in food started on day −2: for groups G4 and G7BIA 5-1058 (30 mg/kg/day), for group G2 bosentan (300 mg/kg/day), forgroup 3 bosentan (100 mg/kg/day), for group 5 BIA 5-1058+bosentan(30+100 mg/kg/day). The remaining animals (groups GI and G6) were givennormal rat chow food. Forty-eight hours later, on day zero, animals fromgroups GI through G5 were administered MCT subcutaneously (60 mg/kg/3ml). Groups G6 and G7 were given vehicle instead of MCT. Treatment wasprolonged for 4 weeks.

MCT was dissolved at 300 mg/kg in 1 N HCl, neutralized with 1 N NaOH anddiluted to 20 mg/ml with sterile water.

Food, water consumption and animal weight was monitored over the allexperiment. Water and food consumption was measured for each cage twicea week. The weights of the individual animals were determined twice aweek.

Animals were kept under treatment for 28 days after MCT administration.The animals that survived were, at that time, anaesthetized withpentobarbital 60 mg/kg administered intraperitoneally, and subjected totissue and organ collection.

Whole spleen, whole liver, both kidneys and lungs were removed, cleanedand weighed. Heart was removed, cleaned from atria and vascular tissueand weighed. Right ventricle was then dissected from the rest of theheart (left ventricle+septum) and both were weighed. Femurs wereremoved, cleaned and length measured with a calliper.

The ratios organ weight to body weight and organ weight to femur lengthwere calculated as well as the ratio right ventricular weight to leftventricular+septum weight.

Irwin Test

The Irwin test (Irwin S. Psycopharmacologia 1968:13; 222-57) is asystematic observational procedure for assessing and scoring thebehavioral and physiological state of rodents. Animals are observedaccording to a standardised observation battery in order to detectneurobehavioural, neurovegetative or psychotropic signs or neurotoxiceffects. A total of 30 parameters are scored using a standardizedprocedure based on that described by Irwin, 1968. These parameters aredistributed as followed: 16 items for the behavioral profile, 9 itemsfor the neurological profile and 5 items for the autonomic profile asrepresented in Table 1 below:

TABLE 1 Behavioural Spontaneous activity locomotor activity spatiallocomotion stereotyped behaviour writhing Motor-affective responsestransfer arousal (appearance) touch response Provoked bitingvocalisation easy of handling/passivity Sensoro-motor visual placingreflex responses pain response (tail pinch) pain response (toe pinch)startle response Posture straub tail abnormal limb position abnormalbody carriage (posture) Neurologic Muscle tone body tone abdominal tonuslimb muscle tonus grip strength Equillibrium and gait righting reflexabnormal gait catalepsy CNS excitability tremors/twitches/jerksconvulsions Autonomic Eyes ptosis Secretions lacrimation salivationGeneral hypothermia piloerection

Results

Survival curves (see FIGS. 2 and 3) clearly show that the combination ofCompound B (30 mg/kg/day) plus bosentan (100 mg/kg/day) significantly(P<0.05) increased the number of animals that survived the 28 daysobserving period when compared to the MCT group. A slight increase insurvival was also observed for the MCT+Compound B group translated by ashift to the right in the survival curve. No clear benefit was observedwith either bosentan treatments, 300 or 100 mg/kg/day as compared to theMCT group.

Thus, the use of Compound B alone resulted in greater survival ratescompared to the use of bosentan alone. Further, the combined use ofCompound B and bosentan resulted in a synergistic effect on survivalrates.

No animals died on the SHAM groups.

As shown in FIGS. 4a, 4b, 5a and 5b , Compound B alone or in combinationwith bosentan significantly decreased the heart, RV and Fulton Index(where the Fulton Index=the ratio of right ventricle weight to leftventricle plus septum weight, i.e. RV/LV+S) as compared to the MCTgroup; the magnitude of the decrease was higher with the combinationCompound B+Bos100 than with the other treatments. Bosentan 300 mg/kg/daysignificantly decreased the RV and Fulton Index as compared to the MCTgroup.

All groups administered with MCT had significantly increased heart, RV,LV+S and Fulton Index as compared to SHAM and SHAM+Compound B groups.

Regarding cardiac remodeling a marked and statistically significantdecrease in right ventricular hypertrophy was observed in theMCT+Compound B+Bos100 group as compared to the MCT group, similarly towhat observed in the MCT+Bos300 group. A significant decreased rightventricular hypertrophy was also observed in the MCT+Bos100 andMCT+Compound B groups.

Thus, Compound B, whether used alone or in combination with bosentan,exhibits beneficial effects in relation to lung injury, particularlypulmonary arterial hypertension.

SYNTHETIC EXAMPLES Example 1(R)-5-aminomethyl-1-(6,8-difluorochroman-3-yl)-1,3-dihydroimidazole-2-thioneHydrochloride

A stirred mixture of (R)-6,8-difluorochroman-3-ylamine hydrochloride(0.22 g, 1.0 mmol),[3-(tert-butyldimethylsilanyloxy)-2-oxopropyl]carbamic acid tert-butylester (0.33 g, 1.1 mmol), potassium thiocyanate (0.11 g, 1.1 mmol) andacetic acid (0.3 mL, 5.0 mmol) in ethyl acetate (3 mL) was refluxed for2 hours, cooled to room temperature, then washed by sodium bicarbonatesolution, dried over anhydrous magnesium sulphate and evaporated invacuo. The residue was purified by the column chromatography over silicagel using ethyl acetate-petroleum ether mixture as eluent. The resultingoil (0.23 g) was dissolved in ethyl acetate (2 ml), whereupon 2M HClsolution in ethyl acetate was added (2 mL, 4 mmol) and the mixture wasstirred for 2 hours at room temperature. The precipitate was removed byfiltration and washed with ethyl acetate to give crystals of m.p. 192°C. (decomp.).

Example 2(R)-5-(2-(benzylamino)ethyl)-1-(6,8-difluorochroman-3-yl)-1H-imidazole-2(3H)-thione

To(R)-5-(2-aminoethyl)-1-(6,8-difluorochroman-3-yl)-1,3-dihydroimidazole-2-thione(2.36 g, 7.58 mmol) and benzaldehyde (0.85 ml, 8.34 mmol) in a mixtureof methanol (15 ml), and dichloromethane (15 ml) sodium cyanoborohydride(0.67 g, 10.66 mmol) was added at 20-25° C. in portions. The mixture wasstirred for 64 h, quenched with IN HCl (12 ml) with stirring followed by3N NaOH (12 ml). The mixture was extracted with DCM (100 ml), theorganic phase was washed with brine (50 ml), dried (MgSO₄) andevaporated to dryness. The residue was purified on a silica gel columnwith ethyl acetate and a mixture of ethyl acetate with methanol (9:1) aseluents. Fractions containing the product were collected, evaporatedunder reduced pressure to approx 20 ml then cooled on ice. Theprecipitate was collected, washed with ethyl acetate-petroleum ether(1:1) mixture, dried on air. Yield was 1.25 g (41%), the product havinga mp 188-90° C. (2-propanol-DCM).

It will be appreciated that the invention may be modified within thescope of the appended claims.

What is claimed is:
 1. A pharmaceutical composition comprising acompound of formula I

wherein, R₁, R₂ and R₃ are the same or different and signify hydrogen,halogen, alkyl, alkylaryl, alkyloxy, hydroxy, nitro, amino,alkylcarbonylamino, alkylamino or dialkylamino group; R₄ signifieshydrogen, alkyl, -alkylaryl or -alkylheteroaryl; X signifies CH₂, oxygenatom or sulphur atom; n is 1, 2 or 3, with the proviso that when n is 1,X is not CH₂; and the individual (R)- and (S)-enantiomers or mixtures ofenantiomers and pharmaceutically acceptable salts thereof, incombination with at least one prostacyclin analogue.
 2. Thepharmaceutical composition according to claim 1, wherein the compound offormula I has the formula IA

wherein, R₁, R₂ and R₃ are the same or different and signify hydrogen,halogen, alkyl, alkylaryl, alkyloxy, hydroxy, nitro, amino,alkylcarbonylamino, alkylamino or dialkylamino group; R₄ signifieshydrogen, alkyl or -alkylaryl group; X signifies CH₂, oxygen atom orsulphur atom; and n is 1, 2 or 3, with the proviso that when n is 1, Xis not CH₂.
 3. The pharmaceutical composition according to claim 1,wherein the compound of formula I has the formula IB

wherein R₁, R₂ and R₃ are the same or different and signify hydrogen,halogen, alkyl, nitro, amino, alkylcarbonylamino, alkylamino ordialkylamino group; R₄ signifies -alkyl-aryl or -alkyl-heteroaryl; Xsignifies CH₂, oxygen atom or sulphur atom; and n is 2 or
 3. 4. Thepharmaceutical composition according to claim 1, wherein the compound offormula I is selected from the group consisting of:(S)-5-(2-aminoethyl)-1-(1,2,3,4-tetrahydronaphthalen-2-yl)-1,3-dihydroimidazole-2-thione;(S)-5-(2-aminoethyl)-1-(5,7-difluoro-1,2,3,4-tetrahydronaphthalen-2-yl)-1,3-dihydroimidazole-2-thione;(R)-5-(2-aminoethyl)-1-chroman-3-yl-1,3-dihydroimidazole-2-thione;(R)-5-(2-aminoethyl)-1-(6-hydroxychroman-3-yl)-1,3-dihydroimidazole-2-thione;(R)-5-(2-aminoethyl)-1-(8-hydroxychroman-3-yl)-1,3-dihydroimidazole-2-thione;(R)-5-(2-aminoethyl)-1-(6-methoxychroman-3-yl)-1,3-dihydroimidazole-2-thione;(R)-5-(2-aminoethyl)-1-(8-methoxychroman-3-yl)-1,3-dihydroimidazole-2-thione;(R)-5-(2-aminoethyl)-1-(6-fluorochroman-3-yl)-1,3-dihydroimidazole-2-thione;(R)-5-(2-aminoethyl)-1-(8-fluorochroman-3-yl)-1,3-dihydroimidazole-2-thione;(R)-5-(2-aminoethyl)-1-(6,7-difluorochroman-3-yl)-1,3-dihydroimidazole-2-thione;(R)-5-(2-aminoethyl)-1-(6,8-difluorochroman-3-yl)-1,3-dihydroimidazole-2-thione;(S)-5-(2-aminoethyl)-1-(6,8-difluorochroman-3-yl)-1,3-dihydroimidazole-2-thione;(R)-5-(2-aminoethyl)-1-(6,7,8-trifluorochroman-3-yl)-1,3-dihydroimidazole-2-thione;(R)-5-(2-aminoethyl)-1-(6-chloro-8-methoxychroman-3-yl)-1,3-dihydroimidazole-2-thione;(R)-5-(2-aminoethyl)-1-(6-methoxy-8-chlorochroman-3-yl)-1,3-dihydroimidazole-2-thione;(R)-5-(2-aminoethyl)-1-(6-nitrochroman-3-yl)-1,3-dihydroimidazole-2-thione;(R)-5-(2-aminoethyl)-1-(8-nitrochroman-3-yl)-1,3-dihydroimidazole-2-thione;(R)-5-(2-aminoethyl)-1-[6-(acetylamino)chroman-3-yl]-1,3-dihydroimidazole-2-thione;(R)-5-aminomethyl-1-chroman-3-yl-1,3-dihydroimidazole-2-thione;(R)-5-aminomethyl-1-(6-hydroxychroman-3-yl)-1,3-dihydroimidazole-2-thione;(R)-5-(2-aminoethyl)-1-(6-hydroxy-7-benzylchroman-3-yl)-1,3-dihydroimidazole-2-thione;(R)-5-aminomethyl-1-(6,8-difluorochroman-3-yl)-1,3-dihydroimidazole-2-thione;(R)-5-(3-aminopropyl)-1-(6,8-difluorochroman-3-yl)-1,3-dihydroimidazole-2-thione;(S)-5-(3-aminopropyl)-1-(5,7-difluoro-1,2,3,4-tetrahydronaphthalen-2-yl)-1,3-dihydroimidazole-2-thione;(R,S)-5-(2-aminoethyl)-1-(6-hydroxythiochroman-3-yl)-1,3-dihydroimidazole-2-thione;(R,S)-5-(2-aminoethyl)-1-(6-methoxythiochroman-3-yl)-1,3-dihydroimidazole-2-thione;(R)-5-(2-benzylaminoethyl)-1-(6-methoxychroman-3-yl)-1,3-dihydroimidazole7-2-thione;(R)-5-(2-benzylaminoethyl)-1-(6-hydroxychroman-3-yl)-1,3-dihydroimidazole-2-thione;(R)-1-(6-hydroxychroman-3-yl)-5-(2-methylaminoethyl)-1,3-dihydroimidazole-2-thione;(R)-1-(6,8-difluorochroman-3-yl)-5-(2-methylaminoethyl)-1,3-dihydroimidazole-2-thione;(R)-1-chroman-3-yl-5-(2-methylaminoethyl)-1,3-dihydroimidazole-2-thione;and(R)-5-(2-(benzylamino)ethyl)-1-(6,8-difluorochroman-3-yl)-1H-imidazole-2(3H)-thione.5. The pharmaceutical composition according to claim 1, wherein thecompound of formula I is(R)-5-(2-aminoethyl)-1-(6,8-difluorochroman-3-yl)-1,3-dihydroimidazole-2-thionehydrochloride.
 6. The pharmaceutical composition according to claim 1,wherein the compound of formula I is(R)-5-(2-(benzylamino)ethyl)-1-(6,8-difluorochroman-3-yl)-1H-imidazole-2(3H)-thione.7. The pharmaceutical composition according to claim 6, wherein the doseof(R)-5-(2-(benzylamino)ethyl)-1-(6,8-difluorochroman-3-yl)-1H-imidazole-2(3H)-thioneis from 10 mg/day to 50 mg/day.
 8. The pharmaceutical compositionaccording to claim 1, wherein the prostacyclin analogue is selected fromthe group consisting of epoprostenol, iloprost, treprostinil andberaprost.
 9. The pharmaceutical composition according to claim 1,wherein the prostacyclin analogue is epoprostenol.
 10. Thepharmaceutical composition according to claim 1, wherein theprostacyclin analogue is iloprost.
 11. The pharmaceutical compositionaccording to claim 1, wherein the prostacyclin analogue is treprostinil.12. The pharmaceutical composition according to claim 1, wherein theprostacyclin analogue is beraprost.
 13. The pharmaceutical compositionaccording to claim 1, wherein the composition is in the form of a singledaily dosage.
 14. The pharmaceutical composition according to claim 1,further comprising one or more inert pharmaceutically acceptablecarriers.
 15. The pharmaceutical composition according to claim 1 inunit dosage form.
 16. The pharmaceutical composition according to claim1, wherein the compound of formula I is(R)-5-(2-(benzylamino)ethyl)-1-(6,8-difluorochroman-3-yl)-1H-imidazole-2(3H)-thioneand the prostacyclin analogue is selected from the group consisting ofepoprostenol, iloprost, treprostinil and beraprost.
 17. A method oftreating pulmonary arterial hypertension comprising administering atherapeutically effective amount of a compound of formula I, or apharmaceutically acceptable salt thereof, in combination with at leastone prostacyclin analogue, to a subject in need thereof.
 18. The methodaccording to claim 17, wherein the compound of formula I is(R)-5-(2-aminoethyl)-1-(6,8-difluorochroman-3-yl)-1,3-dihydroimidazole-2-thionehydrochloride.
 19. The method according to claim 17, wherein(R)-5-(2-(benzylamino)ethyl)-1-(6,8-difluorochroman-3-yl)-1H-imidazole-2(3H)-thione.20. The method according to claim 17, wherein the prostacyclin analogueis selected from the group consisting of epoprostenol, iloprost,treprostinil and beraprost.